Bradykinin stimulates glutamate uptake via both B1R and B2R activation in a human retinal pigment epithelial cells.
OBJECTIVE
We were to examine the effect of bradykinin (BK) in the regulation of glutamate transporter and its related signaling molecules in a human retinal pigment epithelial (ARPE) cells, which are important cells to support retina.


METHODS
d-[2,3-(3)H]-aspartate uptake, western immunoblotting, reverse transcription polymerase chain reaction, [(3)H]-arachidonic acid release, and siRNA transfection techniques were used.


RESULTS
BK stimulated glutamate uptake as well as the mRNA expression of excitatory amino acid transporter 4 (EAAT4) and excitatory amino acid carrier 1 (EAAC1), which was blocked by treatment with bradykinin 1 receptor (B1R) and bradykinin 2 receptor (B2R) siRNA, suggesting the role of B1R and B2R in this process. The BK-induced stimulation of glutamate uptake was also blocked by [des-Arg(10)]-HOE 140, a B1R antagonist, and HOE 140, a B2R antagonist, as well as by the tyrosine kinase inhibitors genistein and herbimycin A. In addition, the BK-induced stimulation of glutamate uptake was blocked by treatment with the phospholipase A(2) inhibitors mepacrine and AACOCF(3), the cyclooxygenase (COX) inhibitor indomethacin, and the COX-2 inhibitor Dup 697. Furthermore, the BK-induced increase in COX-2 expression was blocked by the PI-3 kinase inhibitors wortmannin and LY294002, Akt inhibitor, and the protein kinase C (PKC) inhibitors staurosporine and bisindolylmaleimide I, suggesting the role of PI-3 kinase and PKC in this process. BK stimulated Akt activation and the translocation of PKC activation via the activation of B1R and B2R.


CONCLUSIONS
BK stimulates glutamate uptake through a PKC-Akt-COX-2 signaling cascade in ARPE cells.
Lim
Seul-Ki
SK
Bio-therapy Human Resources Center, Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea.
Park
Min-Jung
MJ
Jung
Ho-Kyoung
HK
Park
Ah-Yeon
AY
Kim
Dong-Il
DI
Kim
Jong-Chun
JC
Bae
Chun-Sik
CS
Kim
Kye-Yeop
KY
Yoon
Kyoung-Chul
KC
Han
Ho Jae
HJ
Park
Soo Hyun
SH
D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology*; D018929:Cell Culture Techniques; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000096:biosynthesis; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D043783:Receptor, Bradykinin B1 / Q000096:biosynthesis* / Q000235:genetics; D043782:Receptor, Bradykinin B2 / Q000096:biosynthesis* / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D027322:Amino Acid Transport System X-AG; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D003609:Dactinomycin; D016718:Arachidonic Acid; D001224:Aspartic Acid; D003513:Cycloheximide; D051546:Cyclooxygenase 2; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D001920:Bradykinin